The estrogen hormone has a broad spectrum of effects on tissues in both females and males. Many of these biological effects are positive, including maintenance of bone density, cardiovascular protection, central nervous system (“CNS”) function, and the protection of organ systems from the effects of aging. However, in addition to its positive effects, estrogen also is a potent growth factor in the breast and endometrium that increases the risk of cancer.
Until recently, it was assumed that estrogen binds to a single ER in cells. As discussed below, this simple view changed significantly when a second ER (ER-β) was cloned (with the original ER being renamed ER-α), and when co-factors that modulate the ER response were discovered. Ligands can bind to two different ERs which, in the presence of tissue-specific co-activators and/or co-repressors, bind to an estrogen response element in the regulatory region of genes or to other transcription factors. Given the complexity of ER signaling, along with the tissue-specific expression of ER-α and ER-β and their co-factors, it is now recognized that ER ligands can act as estrogen agonists and antagonists that mimic the positive effects, or block the negative effects, of estrogen in a tissue-specific manner. This has given rise to the discovery of an entirely new class of drugs, referred to as Selective Estrogen Receptor Modulators or SERMs. These drugs have significant potential for the prevention and/or treatment of cancer and osteoporosis, as well as cardiovascular diseases and neurodegenerative diseases such as Alzheimer's disease.
Bone-resorbing diseases, such as osteoporosis, are debilitating conditions which affect a wide population, and to which there is only limited treatment. For example, osteoporosis affects about 50% of women, and about 10% of men, over the age of 50 in the United States. In individuals with osteoporosis, increased loss of bone mass results in fragile bones and, as a result, increased risk of bone fractures. Other bone-resorption diseases, such as Paget's disease and metastatic bone cancer, present similar symptoms.
Bone is a living tissue which contains several different types of cells. In healthy individuals, the amount of bone made by the osteoblastic cells is balanced by the amount of bone removed or resorbed by the osteoclastic cells. In individuals suffering from a bone-resorbing disease, there is an imbalance in the function of these two types of cells. Perhaps the most well known example of such an imbalance is the rapid increase in bone resorption experienced by postmenopausal women. Such accelerated bone lose is attributed to estrogen deficiency associated with menopause. However, the mechanism of how the loss of estrogen results in increased bone resorption has long been debated.
Recently, investigators have suggested that an increase in bone-resorbing cytokines, such as interleukin-1 (“IL-1”) and tumor necrosis factor (“TNF”), may be responsible for postmenopausal bone loss (Kimble et al., J. Biol. Chem. 271:28890–28897, 1996), and that inhibitors of these cytokines can partially diminish bone loss following ovariectomy in rodents (Pacifici, J. Bone Miner Res. 11:1043–1051, 1996). Further, discontinuation of estrogen has been reported to lead to an increase in IL-6 secretion by murine bone marrow and bone cells (Girasole et al., J. Clin. Invest. 89:883–891, 1992; Jilka et al., Science 257:88–91, 1992; Kimble et al., Endocrinology 136:3054–3061, 1995; Passseri et al., Endocrinology 133:822–828, 1993), antibodies against IL-6 can inhibit the increase in osteoclast precursors occurring in estrogen-depleted mice (Girasole et al, supra), and bone loss following ovariectomy does not occur in transgenic mice lacking IL-6 (Poli et al., EMBO J. 13:1189–1196, 1994).
Existing treatments for slowing bone loss generally involves administration of compounds such as estrogen, bisphosphonates, calcitonin, and raloxifene. These compounds, however, are generally used for long-term treatments, and have undesirable side effects. Further, such treatments are typically directed to the activity of mature osteoclasts, rather than reducing their formation. For example, estrogen induces the apoptosis of osteoclasts, while calcitonin causes the osteoclasts to shrink and detach from the surface of the bone (Hughes et al., Nat. Med. 2:1132–1136, 1996; Jilka et al., Exp. Hematol. 23:500–506, 1995). Similarly, bisphosphonates decrease osteoclast activity, change their morphology, and increase the apoptosis of osteoclasts (Parfitt et al., J. Bone Miner Res. 11:150–159, 1996; Suzuki et al., Endocrinology 137:4685–4690, 1996).
Cytokines are also believed to play an important role in a variety of cancers. For example, in the context of prostate cancer, researchers have shown IL-6 to be an autocrine/paracrine growth factor (Seigall et al., Cancer Res. 50:7786, 1999), to enhance survival of tumors (Okamoto et al., Cancer Res. 57:141–146, 1997), and that neutralizing IL-6 antibodies reduce cell proliferation (Okamoto et al., Endocrinology 138:5071–5073, 1997; Borsellino et al., Proc. Annu. Meet. Am. Assoc. Cancer Res. 37:A2801, 1996). Similar results have been reported for IL-6 with regard to multiple myeloma (Martinez-Maza et al., Res. Immunol. 143:764–769, 1992; Kawano et al., Blood 73:517–526, 1989; Zhang et al., Blood 74:11–13, 1989; Garrett et al., Bone 20:515–520, 1997; and Klein et al., Blood 78:1198–12–4, 1991), renal cell carcinoma (Koo et al., Cancer Immunol. 35:97–105, 1992; Tsukamoto et al., J. Urol. 148:1778–1782, 1992; and Weissglas et al., Endocrinology 138:1879–1885, 1997), and cervical carcinoma (Estuce et al., Gynecol. Oncol. 50:15–19, 1993; Tartour et al., Cancer Res. 54:6243–6248, 1994; and Iglesias et al., Am. J. Pathology 146:944–952, 1995).
Furthermore, IL-6 is also believed to be involved in arthritis, particularly in adjuvant-, collagen- and antigen-induced arthritis (Alonzi et al., J. Exp. Med. 187:146–148, 1998; Ohshima et al., Proc. Natl. Acad. Sci. USA 95:8222–8226, 1998; and Leisten et al., Clin. Immunol. Immunopathol 56:108–115, 1990), and anti-IL-6 antibodies have been reported for treatment of arthritis (Wendling et al., J. Rheumatol. 20:259–262, 1993). In addition, estrogen has been shown to induce suppression of experimental autoimmune encephalomyelitis and collagen-induced arthritis in mice (Jansson et al., Neuroimmunol. 53:203–207, 1994).
The cytokine IL-6 has also been shown to be an important factor in inducing the formation of osteoclasts (Girasole et al., supra; Jilka et al. (1992), supra; Jilka et al. (1995), supra; Kimble et al. (1995), supra; Pacifici et al., supra; and Passeri et al., supra). Other investigators have shown that administration of the neutralizing antibody, antisense oligos, or the Sant 5 antagonist against IL-6, reduces the number of osteoclasts in trabecular bone of ovariectomized mice (Devlin et al., J. Bone Miner 13:393–399, 1998; Girasole et al., supra; Jilka et al. (1992), supra; and Schiller et al., Endocrinology 138:4567–4571, 1997), the ability of human giant cells to resorb dentine (Ohsaki et al., Endocrinology 131:2229–2234, 1993; and Reddy et al., J. Bone Min. Res. 9:753–757, 1994), and the formation of osteoclasts in normal human bone marrow culture. It has also been found that estrogen downregulates the IL-6 promoter activity by interactions between the estrogen receptor and the transcription factors NF-κB and C/EBPβ (Stein et al., Mol. Cell Biol. 15:4971–4979, 1995).
Granulocyte-macrophage colony-stimulating factor (“GM-CSF”) has been suggested to play a role in the proliferation of osteoclastic precursor cells. In long term cultures of human or mouse bone marrow cells or peripheral blood cells, GM-CSF promotes the formation of osteoclastic cells (Kurihara et al., Blood 74:1295–1302, 1989; Lorenzo et al., J. Clin. Invest. 80:160–164, 1987; MacDonald et al., J. Bone Miner 1:227–233, 1986; and Shinar et al, Endocrinology 126:1728–1735, 1990). Bone marrow cells isolated from postmenopausal women, or women who discontinued estrogen therapy, expressed higher levels of GM-CSF than cells from premenopausal women (Bismar et al., J. Clin. Endocrinol. Metab. 80:3351–3355, 1995). Expression of GM-CSF has also been shown to be associated with the tissue distribution of bone-resorbing osteoclasts in patients with erosion of orthopedic implants (Al-Saffar et al., Anatomic Pathology 105:628–693, 1996).
As noted above, it had previously been assumed that estrogen binds to a single ER in cells, causing conformational changes that result in release from heat shock proteins and binding of the receptor as a dimer to the so-called estrogen response element in the promoter region of a variety of genes. Further, pharmacologists have generally believed that non-steroidal small molecule ligands compete for binding of estrogen to ER, acting as either antagonists or agonists in each tissue where the estrogen receptor is expressed. Thus, such ligands have traditionally been classified as either pure antagonists or agonists. This is no longer believed to be correct.
Rather, it is now known that estrogen modulates cellular pharmacology through gene expression, and that the estrogen effect is mediated by estrogen receptors. As noted above, there are currently two estrogen receptors, ER-α and ER-β. The effect of estrogen receptor on gene regulation can be mediated by a direct binding of ER to the estrogen response element (ERE)—“classical pathway” (Jeltsch et al., Nucleic Acids Res. 15:1401–1414, 1987; Bodine et al., Endocrinology 139:2048–2057, 1998), binding of ER to other transcription factors such as NF-κB, C/EBP-β or AP-1—“non-classical pathway” (Stein et al., Mol. Cell Biol. 15:4971–4979, 1995; Paech et al., Science 277:1508–1510, 1997; Duan et al., Endocrinology 139:1981–1990, 1998), and through non-genomic effects via extranuclear estrogen receptor signaling that potentially include plasma membrane ER (Nadal, A. et al., Trends in Pharmacological Sciences 22:597–599, 2001; Wyckoff, M. H. et al., J. Biol. Chem. 276: 27071–27076, 2001; Chung, Y-L. et al., Int. J. of Cancer 97:306–312, 2002; Kelly, M. J. et al., Trends Endocrinol. Metab. 10:369–374, 1999; Levin, E. R. et al., Trends Endocrinol. Metab. 10:374–377, 1999).
Progress over the last few years has shown that ER associates with co-activators (e.g., SRC-I, CBP and SRA) and co-repressors (e.g., SMRT and N-CoR), which also modulate the transcriptional activity of ER in a tissue-specific and ligand-specific manner. In such cases, ER interacts with the transcription factors critical for regulation of these genes. Transcription factors known to be modulated in their activity by ER include, for example, AP-1, NF-κB, C/EBP and Sp-1. In addition, orphan nuclear receptors, such as estrogen receptor-related receptors a, b, g (ERR-a, ERR-b, ERR-g), have been identified. Although estradiol does not appear to be a ligand for the ERRs, some SERMs and other traditional ER-ligands have been shown to bind to the receptors with high affinity (Coward, P. et al., Proc. Natl. Acad. Sci. 98:8880–8884, 2001; Lu, D. et al., Cancer Res. 61:6755–6761, 2001; Tremablay, G. B. et al., Endocrinology 142:4572–4575, 2001; Chen, S. et al., J. Biol. Chem. 276:28465–28470, 2001).
Furthermore, ER-α and ER-β have both overlapping and different tissue distributions, as analyzed predominantly by RT-PCR or in-situ hybridization due to a lack of good ER-β antibodies. Some of these results, however, are controversial, which may be attributable to the method used for measuring ER, the species analyzed (rat, mouse, human) and/or the differentiation state of isolated primary cells. Very often tissues express both ER-α and ER-β, but the receptors are localized in different cell types. In addition, some tissues (such as kidney) contain exclusively ER-α, while other tissues (such as uterus, pituitary and epidymis) show a great predominance of ER-α (Couse et al., Endocrinology 138, 4613–4621, 1997; Kuiper et al., Endocrinology 138, 863–870, 1997). In contrast, tissues expressing high levels of ER-β include prostate, testis, ovaries and certain areas of the brain (Brandenberger et al., J. Clin. Endocrinol. Metab. 83, 1025–8, 1998; Enmark et al., J. Clinic. Endocrinol. Metabol. 82, 4258–4265, 1997; Laflamme et al., J. Neurobiol. 36, 357–78, 1998; Sar and Welsch, Endocrinology 140, 963–71, 1999; Shughrue et al., Endocrinology 138, 5649–52, 1997a; Shughrue et al., J. Comp. Neurol. 388, 507–25, 1997b).
The development of ER-α (Korach, Science 266, 1524–1527, 1994) and ER-β (Krege et al., Proc. Natl. Acad. Sci. USA 95, 15677–82, 1998) knockout mice further demonstrate that ER-β has different functions in different tissues. For example, ER-α knockout mice (male and female) are infertile, females do not display sexual receptivity and males do not have typical male-aggressive behavior (Cooke et al., Biol. Reprod. 59, 470–5, 1998; Das et al., Proc. Natl. Acad. Sci. USA 94, 12786–12791, 1997; Korach, 1994; Ogawa et al., Proc. Natl. Acad. Sci. USA 94, 1476–81, 1997; Rissman et al., Endocrinology 138, 507–10, 1997a; Rissman et al., Horm. Behav. 31, 232–243, 1997b). Further, the brains of these animals still respond to estrogen in a pattern that is similar to that of wild-type animals (Shughrue et al., Proc. Natl. Acad. Sci. USA 94, 11008–12, 1997c), and estrogen still inhibits vascular injury caused by mechanical damage (Iafrati et al., Nature Med. 3, 545–8, 1997). In contrast, mice lacking the ER-β develop normally, are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice (Krege et al., 1998), have normal breast development and lactate normally. The reduction in fertility is believed to be the result of reduced ovarian efficiency, and ER-β is the predominant form of ER in the ovary, being localized in the granulosa cells of maturing follicles.
In summary, compounds which serve as estrogen antagonists or agonists have long been recognized for their significant pharmaceutical utility in the treatment of a wide variety of estrogen-related conditions, including conditions related to the brain, bone, cardiovascular system, skin, hair follicles, immune system, bladder and prostate (Barkhem et al., Mol. Pharmacol. 54, 105–12, 1998; Farhat et al., FASEB J. 10, 615–624, 1996; Gustafsson, Chem. Biol. 2, 508–11, 1998; Sun et al., 1999; Tremblay et al., Endocrinology 139, 111–118, 1998; Turner et al., Endocrinology 139, 3712–20, 1998). In addition, a variety of breast and non-breast cancer cells have been described to express ER, and serve as the target tissue for specific estrogen antagonists (Brandenberger et al., 1998; Clinton and Hua, Crit. Rev. Oncol. Hematol. 25, 1–9, 1997; Hata et al., Oncology 55 Suppl 1, 35–44, 1998; Rohlff et al., Prostate 37, 51–9, 1998; Simpson et al., J Steroid Biochem Mol Biol 64, 137–45, 1998; Yamashita et al., Oncology 55 Suppl 1, 17–22, 1998).
In recent years a number of both steroidal and nonsteroidal compounds which interact with ER have been developed. For example, Tamoxifen was originally developed as an anti-estrogen and used for the treatment of breast cancer, but more recently has been found to act as a partial estrogen agonist in the uterus, bone and cardiovascular system. Raloxifene is another compound that has been proposed as a SERM, and has been approved for treatment of osteoporosis.
Analogs of Raloxifene have also been reported (Grese et al., J. Med. Chem. 40:146–167, 1997).
As for coumarin-based compounds, a number of structures have been proposed, including the following: U.S. Pat. Nos. 6,291,456, 6,331,562 and 6,593,322; Roa et al., Synthesis 887–888, 1981; Buu-Hoi et al., J. Org. Chem. 19:1548–1552, 1954; Gupta et al., Indian J. Exp. Biol. 23:638–640, 1985; Published PCT Application No. WO 96/31206; Verma et al., Indian J. Chem. 32B:239–243, 1993; Lednicer et al., J. Med. Chem. 8:725–726, 1965; Micheli et al., Steroids 5:321–335, 1962; Brandt et al., Int. J. Quantum Chemistry: Quantum Biol. Symposia 13:155–165, 1986; Wani et al., J. Med. Chem. 18:982–985, 1975; Pollard et al., Steroids 11:897–907, 1968.
Accordingly, there is a need in the art for improved compounds useful for treating or preventing a bone-resorbing disease, a neoplastic disease, arthritis, a disease exacerbated by the presence of estrogen or a disease improved by the presence of estrogen; activating the function of ER in a bone cell; inhibiting the function of ER in a cancer cell; inhibiting the expression of IL-6 in a cell; and inhibiting the growth of a neoplastic cell.
Citation or identification of any reference in Section 2 of this application is not to be construed as an admission that the reference is prior art to the present application.